A Case with NAD(P)HX Dehydratase (NAXD) Deficiency: A Newly Defined Mutation in a Novel Neurodegenerative Disorder

Abstract

Introduction

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are required redox equivalents for essential biochemical reactions. Their hydrated forms, NADHX and NAD(P)HX, are inhibitors for several dehydrogenases and cause harmful byproducts. NAD(P)HX dehydratase (NAXD) and NAD(P)HX epimerase (NAXE) together form the nicotinamide repair system.

Case Presentation

A 7-month-old boy was admitted due to myoclonic seizures, impaired consciousness, and rapid loss of head control. One of his siblings regressed after a febrile seizure and died at 7 months. He had lethargy and axial hypotonia but skin lesions and organomegaly were not noted. Basal metabolic tests were within normal limits except serum and cerebrospinal fluid lactate levels, which were mildly elevated. Mitochondrial cocktail was added to the antiepileptic treatment with suspicion of mitochondrial disease. Whole-exome sequencing showed a novel homozygous mutation (c.247G>A) in the NAXD gene. His seizures stopped within a few weeks. However, he died at the age of 18 months.

Discussion

Prominent features of NAXD deficiency are progressive neurological deterioration after fever, cardiomyopathy, skin lesions, and premature death. Unlike the cases reported in the literature, our patient had neither preceding fever nor skin lesion during follow-up. It appears that cases show phenotypic diversity.

Established Facts

• The prominent features of NAD(P)HX dehydratase deficiency are progressive neurological deterioration after fever, cardiomyopathy, skin lesions, and premature death.

Novel Insights

• Seizures may also be the leading clinical finding.

• The disease can also be seen without skin lesions and prior febrile disease.

• Mitochondrial cocktail may be useful in treatment.

Introduction

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are required redox equivalents for essential biochemical reactions, especially in energy metabolism. NADP and its reduced form NAD(P)H are involved in anabolic reactions and cellular oxidative stress protection, while NAD often participates in catabolic reactions. The nicotinamide ring in these cofactors tends to be hydrated and form NADHX and NAD(P)HX in the form of R or S epimers, which can irreversibly transform to cyclic NAD(P)HX enzymatically or spontaneously under stress like acidic pH or high temperature.

NADHX and NAD(P)HX are inhibitors of several dehydrogenases and cause harmful byproducts [Yoshida and Dave, 1975]. The nicotinamide nucleotide repair system eliminates these accumulating byproducts to prevent a toxic environment. There is no repair path for cyclic NAD(P)HX, but NAD(P)HX epimers are converted to NAD(P)H by ATP-dependent enzymes named NAD(P)HX dehydratase (NAXD, MIM *615910) and NAD(P)HX epimerase (NAXE, MIM *608862) [Marbaix et al., 2011]. While the S epimer can be converted directly to NAD(P)H by NAXD, the R epimer must first be converted to S epimer by NAXE and then converted to NAD(P)H and eliminated.

Considering the energy consumption in the brain and the importance of these enzymes, possible damage in their absence may be remarkable. Thus, recently deficiencies of NAXE and NAXD were associated with neurological deterioration episodes exacerbated by fever, and a limited number of cases were reported in the literature so far [Kremer et al., 2016; Spiegel et al., 2016; Van Bergen et al., 2019; Incecik and Ceylaner, 2020]. Here, we report an infant presenting with neurological deterioration without fever and typical skin lesions in whom whole-exome sequencing (WES) demonstrated a novel homozygous mutation in the NAXD gene.

Case Report

A 7-month-old boy was admitted to our clinic due to myoclonic seizures, impaired consciousness, and loss of head control. His seizures had started a few weeks before and continued despite being treated with phenobarbital, pyridoxine, and carbamazepine. Then he quickly lost interest in his surroundings and the head control he had gained when he was 3 months old. There was no previous history of infection or fever. He was born to healthy parents who were first-degree cousins. His birth and postnatal development history were unremarkable but one of his siblings had normal neurodevelopment for up to 2 months, regressed after febrile seizure, and died at 7 months. Upon admission he had lethargy, axial hypotonia, and normal deep tendon reflexes. His head circumference was 44 cm (50th percentile), height was 70 cm (75th percentile), and weight was 9,100 g (75th percentile). Skin lesions and organomegaly were not noted. Eye fundus examination and echocardiography were normal.

When he was admitted to our clinic, his complete blood count, serum biochemistry, C-reactive protein, ammonia, acyl carnitine profile, plasma amino acids, biotinidase activity, urine organic acids, cerebrospinal glucose, and protein levels were within normal limits. Serum and cerebrospinal fluid lactate levels were measured as 2.7 and 1.6 (0.4–1.4) mmol/L. Visual and brainstem auditory evoked potentials were evaluated as normal. Electroencephalography (EEG) revealed epileptiform activity originating from the posterior regions of the bilateral hemispheres with abnormal background activity (Fig. 1). While spinal magnetic resonance imaging (MRI) was normal, cranial MRI showed global cerebral atrophy and corpus callosum hypoplasia (Fig. 1). On magnetic resonance spectroscopy, no lactate peak was detected. Audiological examination stated bilateral conductive hearing loss.

Fig. 1.

a Electroencephalography. Abnormal background and epileptiform activity in occipital regions. b–d Cranial magnetic resonance imaging findings. Axial (b) and sagittal (c) T2 weighted images show mild cerebral atrophy and thin corpus callosum. No diffusion restriction is detected (d).

Carbamazepine was replaced with levetiracetam. Midazolam infusion followed by clobazam and topiramate were initiated upon continuation of myoclonic seizures. When the midazolam dosage was increased, the patient required mechanical ventilation and was extubated after 3 weeks. During the further course, on the 25th day of his hospitalization, mitochondrial cocktail (L-carnitine, coenzyme Q10, vitamin E, thiamine, riboflavin) was added to the treatment with suspicion of mitochondrial disease.

The sequencing analyses of ALDH7A1, MOCS1/2, and POLG genes were normal. WES analysis was performed and revealed a homozygous c.247G>A variant in the NAXD gene. This variant was not previously reported in the literature. Segregation analysis was performed because family history and clinical findings were compatible, and the NAXD c.247G>A variant was found to be heterozygous in the parents.

The NAXD c.247G>A (NM_001242882.1, p.A83T) variant was evaluated according to the American College of Medical Genetics and Genomics (ACMG) criteria [Richards et al., 2015]. The NAXD c.247G>A variant was classified as pathogenic because it was not found in gnomAD genomes, the pathogenic computational verdict based on 9 pathogenic predictions from BayesDel_addAF, DANN, EIGEN, FATHMM-MKL, LIST-S2, M-CAP, MutationAssessor, MutationTaster, and SIFT versus 2 benign predictions from MVP and PrimateAI. The Z -score of the NAXD c.247G>A variant was 0.895 (greater than 0.647).

The patient, whose seizures decreased, was discharged after 1.5 months. EEG at discharge showed diffuse slow waves. Antiepileptic treatment and mitochondrial cocktail continued after the patient was discharged. He was hospitalized one more time during follow-up at the age of 1 year due to pneumonia. Although his condition was rather stable, his neuromotor development had unfortunately stopped completely. He lost his ability to hold his head upright. He died at the age of 18 months because of viral pneumonia and respiratory failure.

Discussion

Although the nicotinamide nucleotide repair system is found in many species, the phenotypic spectrum that may occur in case of its deficiency in humans was only reported in recent years through emerging genetic techniques such as WES or whole-genome sequencing.

Van Bergen et al. [2019] reported increased R-S epimers and cyclic NAD(P)X levels in fibroblasts of affected individuals. Mitochondrial respiratory chain functions were impaired. The levels of thermolability in different variants were distinct. The prominent features in these cases and those reported consecutively were progressive neurological deterioration after fever, cardiomyopathy, skin lesions, and death in the early years of life (Table 1) [Van Bergen et al., 2019; Malik et al., 2020; Zhou et al., 2020]. The accumulated toxic cofactors were thought to cause detachment of the epidermis from dermis [Malik et al., 2020; Zhou et al., 2020]. Skin lesions were observed especially in areas such as flexural creases and posterior neck where the surface temperature is relatively higher [Malik et al., 2020]. Unlike most cases in the literature, our patient had no history of febrile illness when he was first admitted to our clinic, but a rapid neurological deterioration and resistant seizures were evident during his first hospitalization. Of course, a mild fever may have been overlooked by his parents, but his acute phase reactants were also negative at admission. So, we could not prove the presence of any infection. However, the other sibling who had passed away had a history of febrile illness before his deterioration. Post-discharge pneumonias may have contributed to the worsening of his clinical features. We started mitochondrial cocktail quickly with suspicion of mitochondrial disease due to sudden onset myoclonic seizures, EEG findings, and high lactate levels. No skin lesion developed during his whole follow-up. Van Bergen et al. [2019] also reported cases without skin involvement in their study, but all their cases had fever prior to deterioration. The seizures in our case were persistent. Seizures may also be the leading clinical finding in NAXD deficiency. Basal ganglia and white matter-gray matter involvement, which were previously reported, were not seen in this patient, but only cerebral atrophy and thin corpus callosum were detected [Van Bergen et al., 2019].

Table 1.

Clinical features of patients with NAXD mutations

Clinical features	Present case	Van Bergen et al., 2019 (n = 6)	Malik et al., 2020 (n = 1)	Zhou et al., 2020 (n = 1)
Female/male	−/1	4/2	−/1	−/1
Age at onset, months	7	3–43	11	34
Fever prior to deterioration	−	6	+	+
Skin lesion	−	4	+	+
Seizure	+	3	+	−
Lesions in MRI	+	5	N/A	+
EEG	Abnormal background, epileptiform activity in occipital region	Bilateral epileptiform activity Hypsarrhythmia Cerebral dysfunction	N/A	Slow waves in occipital region
NAXD variants NM_001242882.1	c.247G>A (novel mutation)	c.839+1G>T/c.922C>T c.187G>A/c.948_949insTT c.51_54delAGAA c.308C>T c.54_57delAAGA c.331C>T	N/A	c.101_102delTA/c.318C4G
Extraneuronal symptoms	Mild increase in transaminase	Pancytopenia Cataracts Ventricular hypertrophy Dilated cardiomyopathy Vomiting	Leukopenia	Vomiting
Death before 5 years of age	Yes	6/6	Yes	No
EEG, electroencephalography; MRI, magnetic resonance imaging.

Although there is no definitive treatment for NAXD deficiency yet, Zhou et al. [2020] used mitochondrial cocktail and high-dose vitamin B3 in their case, and they saw improvement in the patient's skin lesions without any side effect and no further neurological decline. Since this system converts NAD(P)HX to NAD(P)H, dysfunction in this disease may result in a lack of NAD(P)H, the active form of nicotinic acid. Neurological involvement and skin findings are similar to Pellagra, which is a chronic deficiency of nicotinic acid, and vitamin B3 supplementation was considered to be of benefit [Ying, 2008]. This case has the longest survival with NAXD deficiency in the literature. The authors speculated in their study that the mitochondrial cocktail could be beneficial [Zhou et al., 2020]. We strongly support this approach. After showing deterioration, our case lived for 1 year, but his brother with a similar clinical picture who did not receive mitochondrial cocktail died when he was 7 months old. We suggest that mitochondrial cocktail replacement might be a potential treatment option if NAXD deficiency is suspected even without skin involvement. It may also be more beneficial to start treatment as early as possible.

Conclusion

Although only a limited number of cases have been published so far, it appears that the cases show phenotypic diversity. There is a need for further functional studies about this novel disease. A multidisciplinary approach is very important for adequate treatment of NAXD deficiency, which is a novel and fatal multisystem disease. It should be noted that seizures can more rarely be the main symptom like in our case, and the patient may benefit from mitochondrial cocktail.

Statement of Ethics

Ethical approval was not required for this study in accordance with local/national guidelines. Written informed consent was obtained from the parents of the patient for publication of the details of their medical case and any accompanying images.

Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

Funding Sources

No specific funding was obtained from any agency or organization for the current study.

Author Contributions

G.OT., S.A., and A.A. managed the patient, drafted the article, and revised the final version. N.C.R. performed sequence analysis. All authors read and approved the final manuscript.

Data Availability Statement

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.